Field of the Invention
The invention concerns a process for the production of a hybridoma and a monoclonal antibody obtained therefrom, able to recognize one epitope present on more than one antigen. In particular, the invention relates to a process for the production of a hybridoma and a monoclonal antibody, or fragments thereof, able to recognize more than one vitamin D metabolites, namely 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.
Description of Related Art
Vitamin D is the generic term used to designate vitamin D2 or ergocalciferol and vitamin D3 or cholecalciferol. Humans naturally produce vitamin D3 when the skin is exposed to ultraviolet sun rays. Vitamin D3 is transferred to the liver, where it is metabolised into 25-hydroxyvitamin D3, which is the main form of vitamin D circulating in the body. Since the nineteenth century, vitamin D2 has been available orally through food in order to compensate for a lack of vitamin D3 for example among people who are hardly exposed to sunlight. The oral consumption of vitamin D2 has become increasingly important over recent centuries. In fact, it is currently known that vitamin D has a primary role in the body for calcium binding, and mineralization of bones. It also plays a significant role in various metabolic pathways. The 25-hydroxyvitamin D, and particularly 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 are the forms of vitamin D which are most easily accumulated in the body. These two precursors can be converted by the kidneys to form 1α,25-dihydroxyvitamin D, which is the biologically active form. 1,25-dihydroxyvitamin D relates to the active forms of vitamin D (also known as D hormones) which have a hydroxyl grouping in positions 1 and 25 of the formula (A) and (B). More than fifty or so different metabolites of vitamin D have been discovered to date. Among these are 24,25-dihydroxyvitamin D and 25,26-dihydroxyvitamin D.
It is important to ensure the content of vitamin D in the body can be measured. However, measuring the content of vitamin D is actually of little value, since vitamin D concentrations fluctuate significantly based on the oral consumption of vitamin D2. The same applies equally as regards the physiologically active forms of vitamin D (1α,25-dihydroxyvitamin D), which are also present in the body in relatively low quantities and fluctuate significantly in comparison to 25-hydroxyvitamin D. For these reasons, the quantification of 25-hydroxyvitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) is a valuable tool to facilitate the overall analysis of vitamin D in an individual. Various methods exist, some of which involve immunology, to determine the presence of 25-hydroxyvitamin D.
Usually, immunological methods involve the use of polyclonal antibodies, or monoclonal antibodies. Polyclonal antibodies are known from a long time. To obtain such polyclonal antibodies, animals (such as rabbits for instance) need to be immunised with a highly purified preparation of an antigen. The immunisation has to be done repeatedly with such purified preparation. However, most frequently, this leads to the production of a mixture of antibodies, this mixture randomly binding more than one antigen, thus leading to unspecific measurement. A first issue is the life span of the host animal, Frequently, multiple animals needed to be immunised because of biological variability, Some animals being the source of the most specific antisera have to be selected and the death of said animals lead to the end of the production of the desired antibodies, until next immunisation of animals. Secondly, the antiserum reactivity is also a concern because the desired antibody is only a fraction of the total antibodies in the serum, which is itself a too heterogenic mixture. Further, the technique suffers from batch-to-batch variability. However, the production of polyclonal antibodies is a quick and inexpensive technique which has been and is still widely used in immunology.
In the field of recognition of vitamin D metabolites, a lot of documents describing the use of polyclonal antibodies are known. For instance, Hollis et al. (Clinical chemistry, 1993, 39, 529-533), EP 0092004, Kobayashi et al. (Steroids, 1994, 59, 401-411), and Clemens et al. (Steroids, 1983, 42, 503-509) disclose the production of polyclonal antibodies and their use in vitamin D radioimmunoassay.
In WO 2007/039193, Roche Diagnostics GmbH and F. HOFFMANN-LA ROCHE AG describe various methods of the state of the art. The latter disclose a process to produce polyclonal antibodies against 25-hydroxyvitamin D, which includes steps of immunising an animal with a conjugate which contains 25-hydroxyvitamin D3 or 25-hydroxyvitamin D2 as a hapten, isolating the serum or plasma of this animal and purifying the antibodies contained in the serum or the plasma by immunosorption on a complementary matrix, which includes 25-hydroxyvitamin D2 when the hapten is 25-hydroxyvitamin D3 or which includes 25-hydroxyvitamin D3 when the hapten is 25-hydroxyvitamin D2. EAH-Sepharose has been used as the preferred material for the immunosorption matrix. One major problem arising with this method is that the antibodies are polyclonal, namely that new immunisations must be performed on an animal to produce them. Another problem is that if the method does indeed allow both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 to be determined in a sample, it is necessary to carry out two test batteries involving polyclonal antibodies to find the total amount of the two components of 25-hydroxyvitamin D, i.e. one test to determine the amount of 25-hydroxyvitamin D2 in the sample and another one to determine the amount of 25-hydroxyvitamin D3 in the same sample.
For the quantification of a molecule in biological samples, for instance in diagnostic kits, monoclonal antibodies can also be used. The Kohler and Milstein's invention dated 1975 opened a totally new field in immunology through a technique involving the fusion, in the presence of polyethylene glycol, of a myeloma cell rendered drug sensitive through a mutation in gene HGPRT with immune spleen B cells from a host animal immunised with the antigen of interest. Hybridoma cells survive and may be cultured in an appropriate medium (HAT) and rendered immortal. Because each hybridoma descends from one B cell, it makes copies of only one monoclonal antibody. The hybridoma that produces the antibody of interest is grown in culture to produce large amounts of monoclonal antibody, which are then isolated for further use. It is worth noting that monoclonal antibody is known to be highly specific antibodies against one epitope.
In the field of recognition of metabolites of vitamin D, monoclonal antibodies are used too. For instance Perry et al. (Biochemical and biophysical research communications, 1983, 112, 431-436), U.S. Pat. No. 4,585,741 and Kobayashi et al. (Biol. Pharm. Bull., 1997, 20(9), 948-953) disclose the use monoclonal antibodies against 1,25 dihydroxycholecalciferol. It is worth noting that the normal production scheme of a monoclonal antibody directed against one specific antigen includes the use of one hapten, the immunisation of one animal (generally a mouse), that after fusion between spleen cell and myeloma cell, one hybridoma is produced, and this latter may be cultured and immortalized to produce one monoclonal antibody specifically directed against a single epitope on a single antigen.
If one skilled in the art would like to use monoclonal antibodies for the detection of more than one antigen, he must produce in two parallel processes a monoclonal antibody for each molecule to be detected. For example WO 03/104820 discloses the quantification of vitamins A and D3 in fluid samples. A monoclonal antibody against vitamin A is produced using standard procedures from vitamin A-KLH conjugate (i.e. the hapten is vitamin A). A monoclonal antibody against vitamin D3 is produced from vitamin D3-KLH conjugate (i.e. the hapten is vitamin D3). Hence, two tests have to be performed to obtain the amount of both vitamins in the fluid samples.